Protective role of 17β-estradiol in alcohol-associated liver fibrosis is mediated by suppression of integrin signaling

Background Alcohol-associated liver disease is a complex disease regulated by genetic and environmental factors such as diet and sex. The combination of high-fat diet and alcohol consumption has synergistic effects on liver disease progression. Female sex hormones are known to protect females from liver disease induced by high-fat diet. In contrast, they promote alcohol-mediated liver injury. We aimed to define the role of female sex hormones on liver disease induced by a combination of high-fat diet and alcohol. Methods: Wild-type and protein arginine methyltransferase (Prmt)6 knockout female mice were subjected to gonadectomy (ovariectomy, OVX) or sham surgeries and then fed western diet and alcohol in the drinking water. Results: We found that female sex hormones protected mice from western diet/alcohol-induced weight gain, liver steatosis, injury, and fibrosis. Our data suggest that these changes are, in part, mediated by estrogen-mediated induction of arginine methyltransferase PRMT6. Liver proteome changes induced by OVX strongly correlated with changes induced by Prmt6 knockout. Using Prmt6 knockout mice, we confirmed that OVX-mediated weight gain, steatosis, and injury are PRMT6 dependent, while OVX-induced liver fibrosis is PRMT6 independent. Proteomic and gene expression analyses revealed that estrogen signaling suppressed the expression of several components of the integrin pathway, thus reducing integrin-mediated proinflammatory (Tnf, Il6) and profibrotic (Tgfb1, Col1a1) gene expression independent of PRMT6 levels. Integrin signaling inhibition using Arg-Gly-Asp peptides reduced proinflammatory and profibrotic gene expression in mice, suggesting that integrin suppression by estrogen is protective against fibrosis development. Conclusions: Taken together, estrogen signaling protects mice from liver disease induced by a combination of alcohol and high-fat diet through upregulation of Prmt6 and suppression of integrin signaling.


INTRODUCTION
Alcohol-associated liver disease (ALD) is a complex disease regulated by a combination of genetic and environmental factors.Sex is an important variable, and it is known that both male and female sex hormone signaling regulates liver disease development.In females, there are 3 types of the naturally produced sex hormone, estrogen: 17β-estradiol (E2), estrone, and estriol.17β-estradiol is the predominant circulating estrogen.It signals through estrogen receptor alpha, estrogen receptor beta, and G-protein-coupled estrogen receptor.These receptors are most found in reproductive organs and in the liver, which is one of the most responsive organs to E2 treatment.
The classic mechanism of E2 action involves E2 binding to estrogen receptor alpha or estrogen receptor beta.Estrogen receptor then binds to estrogen response elements.In the liver, genes regulated by estrogen response element are genes involved in lipid metabolism, fatty acid synthesis, cholesterol metabolism, and glucose homeostasis.In addition, estrogen receptor can regulate gene expression indirectly.For example, estrogen receptor alpha interacts with NF-κB, preventing NF-κB from promoting cytokine gene expression. [1,2]strogens play a key role in maintaining liver homeostasis.Lack of estrogen signaling promotes liver fat accumulation and leads to hepatic insulin resistance.Estrogen deficiency exacerbates steatohepatitis in humans and rodent models.Interestingly, in high-fat and high-cholesterol diet in mice, estrogen treatment improved inflammation, liver injury, and serum cholesterol levels. [1,2]In experimental studies, E2 administration protected the liver from hepatic fibrosis by downregulation of the hepatic collagen, hyaluronic acid, and alpha Smooth Muscle Actin expression in male and female rats. [3]Several studies reported the protective role of estrogens in chronic liver disease, and this has been widely accepted and confirmed using gonadectomy in rodent models.However, in a few clinical studies, increased estrogen levels are also implicated in liver disease.This controversy could be due to differences in environmental factors, such as alcohol exposure.
After alcohol exposure, females exhibit injury more quickly than males.Moreover, female livers, when compared with male livers, show increased TLR signaling and NF-κB activity after ethanol treatment, [4,5] suggesting higher sensitivity of female liver to endotoxin/Lipopolysaccharide.In fact, several studies confirm that estrogen treatment increases the sensitivity of hepatic macrophages to endotoxin. [4,6]In addition, estrogen signaling regulates the response of female macrophages to other substances, such as oxysterols, [7] that are crucial for maintaining KC identity and regulating inflammation in the liver. [8][11][12] protein arginine methyltransferase PRMT)6 is an enzyme that catalyzes the formation of mono and asymmetric dimethylarginine.PRMT6 was reported to methylate histones and nonhistone proteins; however, under normal conditions, it has a narrow range of substrates when compared with other methyltransferases, such as PRMT1.In contrast, in the presence of alcohol when PRMT1 is inhibited, [13][14][15][16][17] PRMT6 function becomes critical for maintaining protein methylation. [18]Despite its limited substrate scope, PRMT6 has been reported to regulate a wide range of cellular processes, including cell cycle, cellular senescence, adipocyte differentiation, neuronal function, hematopoietic stem cell differentiation, and many others.Recently, we found that PRMT6 controls liver fibrosis development in alcoholfed mice by regulating integrin methylation. [18]Prmt6 deficiency resulted in a dramatic increase in liver fibrosis development.However, this effect was less pronounced in females.
In this work, we defined the role of female sex hormone signaling in liver disease development induced by a combination of high-fat diet, western diet (WD), and alcohol exposure using a mouse model.We found that female sex hormones protected mice from WD/alcohol-induced liver disease by promoting PRMT6 expression and suppressing integrin gene expression.Loss of female sex hormone signaling resulted in a decrease of PRMT6 expression, and resulting PRMT6dependent liver proteomic changes promoted liver steatosis development.On the other hand, loss of estrogen signaling resulted in an increase of several integrin gene expressions that promoted inflammation and fibrosis in mice fed WD/alcohol.
All mice were housed in a temperature-controlled, specific pathogen-free environment with 12-hour lightdark cycles.All animal handling procedures were approved by the Institutional Animal Care and Use Committee at the University of Kansas Medical Center (Kansas City, KS).The in vivo experiments are reported according to the ARRIVE guidelines.All animal experiments were approved by the KUMC IACUC, as stated in the Methods section.We report strain, age, weight, sex, and detailed procedures as recommended by ARRIVE guidelines.

Gonadectomy
Gonadectomy experiments (ovariectomy, OVX) were performed as described. [20]Females were maintained on a chow diet until 8 weeks of age.At 6 weeks of age, the mice were gonadectomized under isoflurane anesthesia.Ovaries of female mice were removed through an incision just below the rib cage, the muscle layer sutured, and the incision closed with wound clips.In sham-operated control mice, incisions were made and closed as described above.The gonads were briefly manipulated but remained intact.

Tandem mass tag (TMT) labeling and mass spectrometric analysis
Whole liver extracts were lysed in 20 mM HEPES (pH 8.0), 150 mM NaCl, and 0.5% NP-40, 0.1% SDS.Trypsin digestion and TMT labeling were performed following the manufacturer's instructions using the TMTsixplex Mass tagging kit (ThermoFisher Scientific).Briefly, 100 μg of protein per condition were digested with 2.5 µg of trypsin (Promega).After trypsin digestion, TMT label reagent was added.Equal amounts of labeled samples were combined, and the sample was then loaded into a high pH reverse phase spin column previously conditioned following the manufacturer's instructions (ThermoFisher Scientific).The peptides were eluted in 9 fractions.Eluted samples were injected into the HPLC coupled with the Orbitrap Fusion Lumos spectrometer (ThermoFisher Scientific).For data analysis, all tandem mass spectrometry scans were searched using Protein Discoverer v.2.4 running Sequest HT and a mouse database downloaded from the NCBI NR repository.Protein quantification was done utilizing unique peptides only.

Cytokine array
Proteome Profiler Mouse Cytokine Array Kit (R&D Systems) detecting 111 mouse cytokines was used according to the manufacturer's instructions.

Macrophage isolation
Primary peritoneal macrophages were isolated as described. [21]Eight-to 10-week-old mice were killed by CO 2 asphyxiation.Briefly, 10 mL of sterile PBS was injected into the caudal half of the peritoneal cavity using a 25-gage needle (beveled side up), followed by gentle shaking of the entire body for 10 seconds.Saline containing resident peritoneal cells was collected, and cells were plated on uncoated tissue culture plates and incubated for 60 minutes at 37 °C.Nonadherent cells were removed by washing 5 times with warm PBS.Macrophages were maintained in Roswell Park Memorial Institute (RPMI) culture Medium (Invitrogen) containing 10% fetal bovine serum.

Western blotting
Protein extracts (50 µg) were subjected to 10% SDS-PAGE, electrophoretically transferred to nitrocellulose membranes (Amersham Hybond ECL, GE Healthcare), and blocked in 3% BSA/PBS at RT for 1 hour.Primary antibodies were incubated overnight at manufacturerrecommended concentrations.Immunoblots were detected with the ECL Plus Western Blotting Detection System (Amersham Biosciences, Piscataway, NJ) with the ODYSSEY Fc, Dual-Mode Imaging system (Li-COR).

Immunohistochemistry
Liver tissue sections (5-μm thick) were prepared from formalin-fixed, paraffin-embedded samples.The quantification of Sirius Red stained sections was performed in a blinded manner.Immunostaining on formalin-fixed sections was performed by deparaffinization and rehydration, followed by antigen retrieval by heating in a pressure cooker (121 °C) for 5 minutes in 10 mM sodium citrate, pH 6.0, as described.[42] Peroxidase activity was blocked by incubation in 3% hydrogen peroxide for 10 minutes.Sections were rinsed three times in PBS/PBS-T (0.1% Tween-20) and incubated in Dako Protein Block (Dako) at room temperature for 1 hour.After removal of blocking solution, slides were placed into a humidified chamber and incubated overnight with a primary antibody, diluted 1:300 in Dako Protein Block at 4 °C.Ag was detected using the SignalStain Boost immunohistochemistry detection reagent (catalog # 8114; Cell Signaling Technology, Beverly, MA), developed with diaminobenzidine (Dako, Carpinteria, CA), counterstained with hematoxylin (Sigma-Aldrich), and mounted.

RT-PCR
RNA was extracted from livers using the Rneasy Mini Kit (Qiagen).Complementary DNA was generated

Statistics
Student t test was used for the comparison of 2 groups, and ANOVA followed by a post-hoc test for 3 or more groups.When data do not show a normal distribution, the Mann-Whitney U test for 2 groups or the Kruskal-Wallis test for 3 or more groups was used.Multiplicity adjustment was performed with the Bonferroni multiple comparisons test to control the false discoveries when multiple groups are considered.

Female sex hormones protect from liver disease development in alcohol and highfat diet-fed mice
To test the role of female sex hormones in high-fat diet and alcohol-induced liver disease, we fed mice either high-fat WD and plain water as a control or western diet with alcohol (WDA) in the drinking water, as described. [18,19]Female mice underwent gonadectomy (OVX) or sham surgery at 6 weeks of age and were placed on WD control or WDA alcohol diet at 8 weeks of age.After 18 weeks of feeding, we observed that OVX mice gained 60% more weight during WD feeding (Figure 1A, B), and alcohol did not affect the weight gain in agreement with previously described data. [18]Despite differences in weight gain, in both groups (OVX and sham), alcohol induced a comparable increase in liverto-body weight ratios (Figure 1C).We found that OVX  group fed WD with alcohol had significantly elevated serum alanine aminotransferase (ALT) levels compared to sham controls.In contrast, in the WD-only group, there was no difference in ALT levels (Figure 1D), suggesting that in the presence of high-fat diet, female sex hormones were protected from alcohol-induced liver injury.Next, we examined histology and liver fibrosis in 4 groups of mice using Hematoxylin and eosin staining (Figure 1E) and Sirius red staining (Figure 1F).We found that alcohol resulted in a mild liver steatosis in sham mice, which was further elevated in OVX mice (Figure 1E).These changes correlated with increased liver fibrosis in OVX mice fed WD with alcohol compared to sham controls (Figure 1F, G).Taken together, alcohol and OVX surgery additively promoted liver injury, steatosis, and fibrosis.
Elevated liver fibrosis correlated with an increase in inflammation markers such as Tnf (Figure 2D).Since estrogen is known to modulate inflammatory pathways, we examined the circulating cytokine profile in WD-fed and WDA-fed mice using a cytokine array (Figure 2E).We found that in WD-fed mice, OVX resulted in an increase in leptin and a decrease in several chemokines such as CXCL9, CXCL10, and CCL-12 (Figure 2E).In contrast, in the presence of alcohol in addition to the abovementioned changes, OVX promoted an increase in TNFα, IL-17A, CCL-5, RAGE, VEGF, and MMP9, which are known to contribute to liver inflammation and fibrosis.We confirmed that the livers of WDA-fed OVX mice showed higher positive staining for F4/80, and presence of crown-like structures, and inflammatory foci, suggesting that liver inflammation is higher in this group (Figure 2F).

17β-estradiol promotes expression of PRMT6 in the liver
Previously we found that PRMT6, which primarily is expressed in nonparenchymal cells such as liver macrophages, is a key regulator of liver fibrosis development in both high-fat diet and alcohol-fed mice, and loss of PRMT6 promotes fibrosis and steatosis in liver. [23]We observed that females have higher levels of PRMT6 protein (Figure 3A) and mRNA (Figure 3B).These differences are likely due to estrogen signaling since treatment of isolated macrophages with 17βestradiol resulted in a dose-dependent increase in Prmt6 expression (Figure 3C).In agreement with these data, OVX mice had reduced levels in PRMT6 (Figure 3D).
To evaluate the role of PRMT6 in female sex hormone signaling in the liver, we compared proteomic changes in OVX versus sham mice and proteomic changes in Prmt6 KO mice compared to wild-type (WT) controls using TMT Mass Spectrometry analysis (Figure 3E). [19,24]This analysis was performed in alcohol-fed mice only.We found a strong positive correlation between these 2, suggesting that OVXinduced proteomic changes could be mediated by the loss of PRMT6.In addition, we were able to quantify the abundance of several arginine-methylated peptides in these mice (Figure 3F).Five of them (NOD-like receptor family pyrin domain containing 6, ARHGAP17, RAB1B, CIMIP4, and SYNE2) showed reduced abundance in KO mice compared to WT controls, suggesting that they could be targets of PRMT6 enzymatic activity.We noted that methylation of these proteins was not affected or affected to a lesser extent by PRMT6 KO in OVX mice (Figure 3F), further confirming that PRMT6 activity is reduced in OVX mice.We next analyzed the gene ontology term enrichment in upregulated and downregulated proteins in OVX mice (Figure 3G).We found that upregulated proteins were enriched for proteins involved in fatty acid metabolism and oxidative stress response, suggesting that these changes could contribute to OVX-induced liver injury and steatosis.

PRMT6 mediates protective effect of female hormone signaling
To assess whether PRMT6 mediates the protective effect of female hormone signaling in the liver, we performed OVX or sham surgeries in Prmt6 KO mice and fed them WD or WD with alcohol for 18 weeks (Figure 4A).We found that in contrast to WT mice, there was no difference in weight gain.All groups gained similar weight, which was comparable to the weight gain of OVX WT mice (Figure 4A compared to Figure 1B).Histologically, we observed that all 4 groups of mice had similar steatosis (Figure 4B) and showed no difference in serum ALT (Figure 4C).To quantitatively assess the liver steatosis phenotype in these mice, we measured (N = 3 per group).The ratio of protein abundance in gonadectomy/sham groups (OVX/Sham) in WT plotted versus ratio of protein abundance in KO/WT sham mice.(F) Fold change in methylated peptide abundances (KO/WT) in mice from gonadectomy (OVX) and Sham groups (G) GO term biological process enrichment analysis in top upregulated and downregulated proteins.Abbreviations: ARHGAP17, Rho GTPase Activating Protein 17; CIMIP4, ciliary microtubule inner protein 4; PRMT6, protein arginine methyltransferase 6; KO, knockout; OVX, ovariectomy; RAB1B, Ras-related protein Rab-1B; SYNE2, Spectrin Repeat Containing Nuclear Envelope Protein 2; TMT, tandem mass tag; WT, wild type.liver TG levels (Figure 4D).We found that in WT mice, OVX and alcohol additively increased TG levels.In contrast, in Prmt6 KO mice, there was no difference between groups (Figure 4D).
To further assess the role of estrogen signaling in liver steatosis in WD and alcohol-fed mice, we treated male mice that were fed WD with alcohol in the drinking water for 20 weeks with exogenous estrogen (17βestradiol, 0.25 mg/kg twice a week starting week 16).We observed that WT male mice treated with estrogen showed slightly reduced weight gain after the beginning of treatment, which did not reach statistical significance (p = 0.1).Prmt6 knockout mice showed no difference in weight gain after estrogen treatment (Figure 4E).We found that estrogen treatment greatly reduced steatosis in male mice (35% reduction in liver TG) without affecting liver-to-body weight ratios (Figure 4F, G).In contrast, in Prmt6 KO mice, estrogen treatment did not significantly affect liver steatosis (18% reduction, p > 0.05, Figure 4F, G).
Taken together, estrogen-mediated PRMT6 upregulation protects female mice from WD/alcohol-induced weight gain and liver steatosis.

Female sex hormones protect from alcohol and WD-induced fibrosis in PRMT6 independent way
We next examined the effect of OVX in Prmt6 KO mice on liver fibrosis by Sirius Red staining (Figure 5A, B).We found that Prmt6 KO in WD-fed female mice promoted liver fibrosis, and alcohol feeding similarly promoted liver fibrosis in WT females.However, in Prmt6 KO mice, alcohol did not produce additional increase in liver fibrosis.Similar results were observed in sham Prmt6 KO mice, which had liver fibrosis levels higher than WT WD controls, but no additional effect of alcohol was observed.In contrast, the OVX mice group showed significantly elevated fibrosis levels after alcohol feeding.
Taken together, in WD-fed WT mice, OVX resulted in a small increase in liver fibrosis (Figures 1 and 2), which is likely due to PRMT6 loss (since the OVX effect is absent in KO WD-fed mice).In contrast, in WD/alcoholfed females, OVX promoted an additional increase in fibrosis independent of PRMT6.
We next evaluated the effect of estrogen treatment on fibrosis in male mice (Figure 5C, D).In male mice fed WD, Prmt6 KO resulted in a small increase in liver fibrosis similarly to female mice.In WD/alcohol-fed males, alcohol further increased liver fibrosis in both WT and KO mice, similarly to OVX females but in contrast to female sham controls.Estrogen treatment reduced liver fibrosis in males abolishing the difference between WT and Prmt6 KO.
Taken together, estrogen suppressed WD/alcoholinduced liver fibrosis development in both male and female mice independent of PRMT6.These effects were not due to altered alcohol intake.We found that OVX females consumed less alcohol than sham controls but had more liver fibrosis.In male mice, we observed the opposite; estrogen-treated mice had slightly higher alcohol consumption (Figure 5E).
We further assessed levels of liver fibrosis and inflammation in these mice (Figure 6A-C).We found that increased liver fibrosis in OVX Prmt6 KO mice fed alcohol correlated with elevated mRNA levels of Col1a1, Tgfb1, and Tnf (Figure 6A, B), similarly to WT OVX mice.We observed that these mice also showed elevated COL1A1 and αSMA staining (Figure 6C).In male mice, WD/alcohol-fed KO mice showed significantly elevated Col1a1 and Tnf mRNA compared to WT controls.Estrogen treatment reduced Col1a1 levels in KO mice to the level of WT controls; however, Tnf levels in KO mice were unchanged (Figure 6D).

Female sex hormones suppress integrin signaling in the liver
To assess the mechanism of female sex hormonemediated liver fibrosis regulation, we examined proteomic changes in OVX KO mice compared to sham control (Figure 6E, F).We found that OVX-induced proteomic changes were different in WT and KO mice (Figure 6E).Gene ontology term enrichment analysis of proteins regulated by OVX in KO mice (Figure 6F) showed that proteins upregulated by OVX in KO mice included several molecules involved in cell adhesion, such as integrins (Figure 6G).
We next examined whether estrogen and female hormone signaling modulated integrin pathway (Figure 7).We found that in isolated macrophages, estrogen treatment suppressed the expression of several integrin genes, including genes for integrins α1, α4, αx, β1, β2, and integrin-linked kinase (Figure 7A).In agreement with these data, integrin gene expression was significantly elevated in OVX mice compared to sham controls (Figure 7B).OVX effect was  present in both WT and KO mice fed WD as well as WD/ alcohol.For some genes such as Itgax OVX, the effect was greater in WDA-fed Prmt6 KO.We tested whether mRNA changes translated into protein changes (Figure 7D).We found that integrin α4 protein expression is undetectable in WD control livers and slightly increased in WD/alcohol mice.In contrast, ITGA4 expression was greatly elevated in OVX livers independent of the diet (Figure 7D).To test if this mechanism is relevant in human disease, we analyzed integrin expression (ITGA4, ITGAX) in human blood monocytes from patients with liver disease (Figure 7E) obtained from KUMC Liver Bank.We found that monocytes from female patients with ALD had lower levels of integrin gene expression compared to male patients.Interestingly, this difference was reversed in patients with HCV.

Female sex hormones protect from alcohol and WD-induced fibrosis and inflammation via integrin signaling
To evaluate the role of estrogen-mediated suppression of integrin signaling, we tested the role of integrin signaling using small hairpin RNA-mediated knockdown in isolated macrophages (Figure 8A).We found that estrogen treatment significantly reduced gene expression of proinflammatory cytokines such as Tnf and Il6.Integrin gene (Itga4 or Itgax) knockdown similarly reduced Tnf and Il6 gene expression, and estrogen treatment had no additional effect in the knockdown cells, suggesting that reduced proinflammatory gene expression in estrogentreated macrophages is mediated by estrogen-mediated integrin suppression.We next tested whether we could mimic these effects using peptides containing Arg-Gly-Asp integrin-binding motif (Figure 8B).In vitro treatment with 2 Arg-Gly-Asp-containing peptides phenocopied the effect of integrin knockdown, peptides reduced proinflammatory gene expression, and abolished the effect of estrogen on macrophages (Figure 8B).
Finally, we tested the effect of integrin inhibition in vivo.Male and female mice were fed WD with alcohol in the drinking water (20% alcohol for 8 weeks) and received twice weekly injections of Arg-Gly-Asp peptide mix (2.5 mg/kg each) during the last 4 weeks of feeding.Peptide injections did not affect weight gain or liver-tobody weight ratios (Figure 8C).We found that peptidetreated mice had significantly lower levels of Col1a1 and Il6 mRNA, suggesting that inflammation and fibrosis were attenuated in these mice (Figure 8D).

DISCUSSION
Alcohol-associated liver disease is a complex disease influenced by both environmental and genetic factors, such as age and sex.[30] Recent single-cell RNA-seq studies confirmed that alcohol effects on every liver cell population were indeed sex specific. [31,32]In human patients, females have higher mortality in acute alcohol-associated hepatitis, even though men have higher serum creatinine, ALT, and GGT concentrations. [25]Estrogen is considered to contribute to increased susceptibility of women to alcohol-associated liver disease. [33,34]This hypothesis is supported mostly by in vitro studies in isolated cells.On the other hand, the role of estrogen in metabolic dysfunction-associated steatotic liver disease (MASLD) progression is considered mostly protective.][37] Rodent gonadectomy experiments in mice suggested that these pathways are, in part, regulated by sex hormone signaling. [35]In humans, lack of estrogen in postmenopausal women is associated with increased susceptibility to MASLD. [1,2]This notion is supported by multiple studies in rodent models of liver disease.
The combination of MASLD with heavy alcohol consumption is termed MetALD (MASLD + alcohol 140-350 g/wk and 210-420 g/week for females and males, respectively).Recent clinical studies examining existing databases such as UK Biobank or National Health and Nutrition Examination Survey (NHANES) data set using this new nomenclature revealed that the MetALD (MASLD with heavy alcohol consumption) group comprises predominantly males (66% in UK Biobank or 71% in NHANES vs. 55%-60% in MASLD controls) and have higher serum ALT and aspartate aminotransferase levels but lower incidence of diabetes and dyslipidemia compared to MASLD. [38,39]However, no systematic assessment of the role of female sex and female sex hormone signaling in MetALD was performed.In this study, we examined the role of female sex hormones and arginine methyltransferase PRMT6 in liver disease development using a combination of high-fat diet (WD) feeding and alcohol in the drinking water.We found that female sex hormones protected mice from WD/alcohol-induced liver disease by promoting PRMT6 expression, which prevented WD-induced weight gain and liver steatosis, and by suppressing integrin gene expression, which protected female mice from alcohol-induced liver fibrosis (Figure 8E).Loss of female sex hormone signaling resulted in increased weight gain, steatosis, and fibrosis in mice fed WD/ alcohol.Interestingly, in control mice fed WD with plain water, loss of female sex hormone signaling also resulted in weight gain, liver steatosis, and mild fibrosis; however, these were less pronounced and fully dependent on PRMT6 (Figure 8E).
Previously, we identified PRMT6 as a key regulator of liver fibrosis development in mice fed high-fat diet, alcohol, or treated with thioacetamide as well as mice on a chow diet at 1 year of age. [18]One of the main protective functions of PRMT6 is macrophage integrin methylation, which reduces their profibrotic function in liver disease induced by multiple factors: high-fat diet, alcohol, thioacetamide treatment, or aging. [23]In this study, we found that female sex hormone signaling and specifically, estrogen, suppresses integrin gene expression.
Thus, PRMT6 function in protecting female mice from alcohol-induced liver fibrosis is reduced, which correlates with observed minor differences in fibrosis levels between female wild type and Prmt6 knockout mice fed alcohol.In contrast, the effect of alcohol and Prmt6 knockout is synergistic in male mice.We demonstrated that in agreement with this hypothesis, estrogen treatment in male mice protected them from liver fibrosis and reduced differences between wild-type and knockout mice.
Our study revealed a complex interplay between female hormone signaling and PRMT6 activity, which is especially evident from our proteomic study.An intriguing finding from that study was that PRMT6 loss and female hormone signaling loss promoted similar changes in liver proteome; in contrast, the combination of both revealed many novel targets of female sex hormone signaling in the liver.Several of these targets were involved in cellular adhesion and integrin signaling.Our data suggests that estrogen-mediated integrin regulation is important factor of ALD development.
Previous studies indicated that PRMT6 has very narrow substrate specificity and methylates very few targets outside the nucleus. [40]Despite the small number of substrates, it regulates multiple processes in the liver, including lipid metabolism, inflammation, and fibrosis development.It is an important regulator in humans as well.Genetic polymorphisms in PRMT6 gene were found to be associated with body weight, cholesterol, and bilirubin levels, as well as serum alanine aminotransferase measurement, suggesting that PRMT6 in humans could be involved in liver function.Previously, we identified that liver PRMT6 levels negatively correlate with liver fibrosis in patients with liver disease. [23]Here, we found that PRMT6 regulates weight gain and liver steatosis, while its effect on fibrosis is sex dependent and mediated by estrogen signaling.Further studies are necessary to test the role of PRMT6 genetic polymorphisms in the susceptibility to liver disease (ALD and/or MetALD) and the role of estrogen, especially in premenopausal and postmenopausal female patients.
Interestingly, PRMT6 genetic polymorphisms were associated in humans with testosterone levels and sex hormone-binding globulin levels in postmenopausal women.This is another aspect that could contribute to sex differences observed in our study and a subject of a future investigation.
One of the limitations of our study is the use of wholebody knockout mice.PRMT6 is primarily expressed in nonparenchymal cells, and its expression in liver macrophages is the highest compared to stellate cells or endothelial cells, 2 other main non-parenchymal cells populations in the liver.It is likely, however, that the weight gain effect reported here could be mediated by extrahepatic PRMT6, and the effect on liver steatosis could be due to PRMT6 expression in hepatocytes.Previously, we have shown that PRMT6 and integrin signaling in liver macrophages is mediating the effects on liver fibrosis development. [23]However, integrindependent mechanisms could be important in other cell types as well.While integrins α4 and αx are enriched in macrophages.[43][44][45] Further studies using cell type-specific knockout mice are necessary to define the role of specific integrins in estrogen-mediated protective effect for LD development.
In conclusion, we showed that female sex hormone signaling and PRMT6 interplay is an important modulator of ALD development through regulation of liver steatosis, liver injury, and fibrosis in the setting of highfat diet feeding, which has important implications for patients with MetALD.Specifically, we found that estrogen increased PRMT6 levels and suppressed liver integrin gene expression and thus prevented fibrosis development.Several integrin target therapies are now in clinical trials for different inflammatory conditions and cancer.Our data suggest that future potential integrintargeted therapy for ALD must consider estrogen and PRMT6 levels (or polymorphism status) to define the candidates who might benefit the most.

1
Female sex hormones protect from alcohol and high-fat diet-induced liver injury and fibrosis.Female wild-type mice were subjected to gonadectomy (OVX) or sham surgery and placed on WD control diet or WD with alcohol in the drinking water (alternating 10% and 20%) for 18 weeks.(A and B) Weight change over time in sham and OVX mice fed WD or WDA from 4 groups.**, p < 0.001 between OVX and sham controls, by linear regression comparison.(C) Liver-to-body weight ratios N = 4-10 mice per group.*, p < 0.05, by one-way ANOVA with Bonferroni multiple comparisons test.(D) Serum ALT at 12 weeks of feeding.N = 3-10 mice per group.**, p < 0.01, by one-way ANOVA.(E).Representative images of H&E staining in mice subjected to sham surgery or gonadectomy (OVX).(F) Representative images of Sirius red staining.(G) Percent positive Sirius Red staining area.N = 4-10 mice per group.*, p < 0.05, by one-way ANOVA with Bonferroni multiple comparisons test.Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; H&E, Hematoxylin and eosin; OVX, ovariectomy; WD, western diet; WDA, western diet with alcohol.

F
I G U R E 3 Female sex hormones promote profound proteomic changes in the liver in PRMT6 dependent way.(A-D) Estrogen promotes PRMT6 expression.(A) Representative images of PRMT6 protein staining in livers of male and female mice fed chow diet.(B) Mice (male and female) were fed WD (control) or WD with alcohol in the drinking water (alcohol).Relative Prmt6 gene expression.N = 5-10 mice per group.*, p < 0.05, **, p < 0.01 by one-way ANOVA with Bonferroni multiple comparisons test.(C) Relative Prmt6 gene expression in mouse peritoneal macrophages treated in vitro with 10 nM, 100 nM, or 1 µM 17β-estradiol solution.N = 4 independent experiments.*, p < 0.05, **, p < 0.01 01 by one-way ANOVA.(D) Representative images of PRMT6 protein staining in mice subjected to sham surgery or gonadectomy (OVX).(E) TMT mass spectrometry analysis of wild type and Prmt6 knockout mice that were subjected to sham or gonadectomy surgeries and fed WD with alcohol PROTECTIVE ROLE OF 17Β-ESTRADIOL IN ALD | 7

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I G U R E 4 Female sex hormones protect from alcohol and high-fat diet-induced steatosis.(A-D) Female Prmt6 knockout mice were subjected to gonadectomy (OVX) or sham surgery and placed on WD control diet or WD with alcohol in the drinking water (alternating 10% and 20%) for 18 weeks.(A) Weight change over time in mice from 4 groups.(B) Liver-to-body weight ratios in KO mice, N = 3-10 mice per group.Right.Representative images of H&E staining from mice subjected to sham surgery or gonadectomy (OVX) fed WD or WDA diet.(C) Serum ALT at 12

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weeks of feeding N = 3-5 mice per group.(D) Liver triglyceride measurement in WT and Prmt6 KO mice.N = 3-10 mice per group.*, p < 0.05, **, p < 0.01 01 by one-way ANOVA.(E-G) Male mice were fed WD with alcohol in the drinking water for 20 weeks.Starting week 16 mice received twice weekly 0.25 mg/kg of 17β-estradiol in PBS, IP. (E) Weight change over time in WT and Prmt6 KO mice injected with estrogen or control (saline).Arrow indicates the start of the treatment.(F) Liver-to-body weight ratios in WT and KO mice.Right.Representative images of H&E staining (G) Liver triglyceride measurement in WT and Prmt6 KO mice.N = 4-8 mice per group.**, p < 0.01 by Student t test.Abbreviations: ALT, alanine aminotransferase; KO, knockout; OVX, ovariectomy; TG, triglycerides; WD, western diet; WDA, western diet with alcohol; WT, wild type.Female sex hormones protect from alcohol and high-fat diet-induced liver injury and fibrosis in a PRMT6-independent way.(A and B) Prmt6 knockout mice were subjected to gonadectomy (OVX) or sham surgery and placed on WD control diet or WD with alcohol in the drinking water (alternating 10 and 20%) for 18 weeks.(A) Representative images of Sirius red staining.(B) Percent positive Sirius Red staining area.N = 3-6 mice per group.*, p < 0.05, **, p < 0.01 by one-way ANOVA with Bonferroni multiple comparisons test.Right.Western blot analysis of αSMA protein levels from 4 groups.(C and D) Male mice were fed WD with alcohol in the drinking water for 20 weeks.Starting week 16 mice received twice weekly 0.25 mg/kg of 17β-estradiol in PBS, IP. (C) Representative images of Sirius red staining.(D) Percent positive Sirius Red staining area.N = 4-8 mice per group.*, p < 0.05, **, p < 0.01 by one-way ANOVA with Bonferroni multiple comparisons test.(E) Alcohol intake in sham control or OVX females (red circles) and control and control (saline) or estrogen-treated male (blue circles) mice.Abbreviations: αSMA, alpha smooth muscle actin; KO, knockout; OVX, ovariectomy; WD, western diet; WDA, western diet with alcohol; WT, wild type.PROTECTIVE ROLE OF 17Β-ESTRADIOL IN ALD I G U R E 6 Female sex hormones protect from alcohol and high-fat diet-induced inflammation and fibrosis in Prmt6 KO mice.(A and B) Prmt6 knockout mice were subjected to gonadectomy (OVX) or sham surgery and placed on WD control diet or WD with alcohol in the drinking water (alternating 10 and 20%) for 18 weeks.(A and B) Whole liver mRNA N = 3-5 mice per group.**, p < 0.01 by Student t test.(C) Representative images of immunohistochemistry staining of liver tissue using COL1A1 or αSMA specific antibodies.N = 3. *, p < 0.05 compared to WD control by student t test.(D) Whole liver mRNA for WT and KO male mice injected with estrogen or control, N = 5-10 mice per group.*, p < 0.05 by student t test.(E) The ratio of protein abundance in gonadectomy/sham groups (OVX/Sham) in WT and KO mice.(F) GO term biological process enrichment analysis in top upregulated and downregulated proteins.(G) Relative protein abundance of cell adhesion molecules in KO mice subjected to sham (Sham) surgery or gonadectomy (OVX).Abbreviations: αSMA, alpha smooth muscle actin; COL1A1, collagen 1A1; GO, gene ontology; KO, knockout; OVX, ovariectomy; WD, western diet; WDA, western diet with alcohol; WT, wild type.

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I G U R E 7 Estrogen suppresses integrin expression.(A) Relative gene expression in mouse peritoneal macrophages treated in vitro with 10 nM, 100 nM, or 1 µM 17β-estradiol solution.N = 3 independent experiments.*, p < 0.05, **, p < 0.01 by paired t test.(B and C) Female wild-type and Prmt6 KO mice were subjected to gonadectomy (OVX) or sham surgery and placed on WD control or WDA alcohol diet for 18 weeks.Whole PROTECTIVE ROLE OF 17Β-ESTRADIOL IN ALD | 13

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I G U R E 8 Estrogen suppresses proinflammatory and profibrotic gene expression via integrins.(A) Relative gene expression in mouse peritoneal macrophages transfected with shRNA specific for Itga4, Itgax, or control shRNA treated in vitro with 100 nM of 17β-estradiol solution (E).N = 4 independent experiments.*, p < 0.05, **, p < 0.01 by paired t test.(B) Relative gene expression in mouse peritoneal macrophages treated in vitro with 100 nM of 17β-estradiol solution (E) in the presence or absence of 10 µg/mL of indicated RGD peptides individually or combined.N = 4 independent experiments.*, p < 0.05, **, p < 0.01 by paired t test.(C and D) Male and female mice were fed WD with alcohol in the drinking water PROTECTIVE ROLE OF 17Β-ESTRADIOL IN ALD | 15 ). Quantitative real-time reverse transcription-polymerase chain reaction was performed in a CFX96 real-time system (Bio-Rad) using specific sense and antisense primers combined with iQ SYBR Green Supermix (Bio-Rad) for 40 amplification cycles: 5 seconds at 95 °C, 10 seconds at 57 °C, and 30 seconds at 72 °C.mRNA concentrations were calculated relative to Actb and normalized to the average expression of the control condition.
liver mRNA.N = 3-10 mice per group.*, p < 0.05, **, p < 0.01 by one-way ANOVA with Bonferroni multiple comparisons test in WT and KO mice separately.(D) Representative images of immunohistochemistry staining in WT female mice using integrin α4 (ITGA4) specific antibody.(E) Relative gene expression in blood monocytes from female and male patients with ALD or HCV *, p < 0.05 by Student t test.Abbreviations: ALD, alcohol-associated liver disease; Ilk, integrin-linked kinase; KO, knockout; OVX, ovariectomy; WD, western diet; WDA, western diet with alcohol; WT, wild type.